芝麻发芽期耐盐性鉴定方法研究及耐盐候选基因的挖掘

Screening Method for Salt Tolerance in Sesame(Sesamum indicum L.) and Identification of Candidate Salt-Tolerant Genes

【目的】确定芝麻发芽期耐盐性鉴定适宜的Na Cl胁迫浓度和评价指标,发掘耐盐种质和耐盐相关重要基因,为芝麻耐盐性大规模鉴定、遗传改良和耐盐机理研究提供方法借鉴和优异基因资源。【方法】以8份耐盐性差异较大的芝麻种质为材料,在不同浓度的Na Cl(0、50、100、150、200和250 mmol·L-1)胁迫下发芽,测定其发芽势、成苗率、根长、芽长和鲜重等指标,通过对指标值的方差分析、主成分分析、隶属函数和相关性分析等,筛选芝麻发芽期耐盐性鉴定适宜的Na Cl处理浓度和评价指标。以100 mmol·L-1 Na Cl溶液为处理浓度,相对成苗率为鉴定指标对71份芝麻核心种质进行发芽期耐盐性鉴定和全基因组关联分析,并对获得的候选基因进行功能注释;通过Na Cl胁迫下芝麻幼苗叶片转录组测序和荧光定量PCR分析候选基因的表达模式,筛选耐盐相关候选基因。【结果】对不同浓度Na Cl胁迫下8份芝麻材料发芽各指标进行统计和方差分析表明,100 mmol·L-1的Na Cl处理下,除发芽势外,发芽指数、活力指数、成苗率、根长、芽长和苗鲜重的标准偏差值均较大,所有指标在0.05水平上均存在显著差异,100 mmol·L-1的Na Cl溶液可以作为芝麻发芽期耐盐性鉴定的适宜胁迫浓度;相对成苗率、相对发芽势、相对发芽指数、相对活力指数、相对芽长、相对根长和相对鲜重7个指标与芝麻发芽期耐盐性关系密切,贡献率较高,可以作为芝麻发芽期耐盐性鉴定的评价指标。71份芝麻核心种质材料的相对成苗率变异比较丰富,符合正态分布;通过对71份芝麻核心种质相对成苗率与SNP标记进行全基因组关联分析,检测到7个与芝麻发芽期耐盐性显著关联的SNP标记(LG5:688003、LG7:9582027、LG10:5274091、LG10:10788493、LG11:11924186、LG14:2128695和LG16:3930301),SNP标记上、下游各100 kb区间内共有基因67个,其中有功能注释的基因34个;通过耐盐材料S04在盐胁迫下的转录组测序和荧光定量PCR对预测的候选基因进行表达模式分析,共有21个候选基因受到盐胁迫诱导显著差异表达。【结论】芝麻发芽期耐盐性鉴定的适宜Na Cl胁迫浓度为100 mmol·L-1,相对成苗率等7个指标可以作为适宜的评价指标;检测到与芝麻发芽期耐盐性显著关联的SNP标记7个,并鉴定出耐盐候选基因21个。

【Objective】In order to evaluate salt tolerance of sesame germplasms and reveal the genetic basis of salt tolerance in sesame at the germination stage, optimum Na Cl concentration and best salt tolerance indexes were determined, salt-tolerant sesame genotypes as well as candidate genes were identified in this study. 【Method】 Eight sesame genotypes with different salt tolerance were germinated under different Na Cl concentrations（0, 50, 100, 150, 200 and 250 mmol·L^-1）. Seven germination indexes including germination potential, germination index, vigor index, seedling rate, radicle length, embryo length and fresh weight of seedling were measured. The optimum Na Cl concentration and salt tolerance indexes of sesame were determined using variance analysis, principal component analysis, membership function and correlation analysis based on the relative values of multiple indexes. In addition, genome-wide association analysis（GWAS） of salt tolerance related trait in 71 sesame germplasms was performed to find the SNP loci and candidate genes related to salt tolerance in sesame. Gene function annotation, transcriptome analyses and real-time quantitative reverse transcriptase PCR（q RT-PCR） were used to identify important candidate genes involved in salt tolerance. 【Result】The larger standard value deviations of germination indexes were observed at 100 mmol·L^-1 Na Cl concentration, implying that 100 mmol·L^-1 is the optimum Na Cl concentration for salt-tolerance screening in this study. The seven indexes were highly correlated with salt tolerance at germination stage, suggesting that, these indexes could be targeted for effective screening of salt-tolerance in large sesame germplasm. The genome-wide association analysis identified 7 SNP loci peaks（LG5：688003, LG7：9582027, LG10：5274091, LG10：10788493, LG11：11924186, LG14：2128695 and LG16：3930301） significantly associated with salt tolerance and 34 functional candidate genes. The transcriptome analysis and q RT-PCR showed that 21 candidate genes respond strongly to salt stress.【Conclusion】In this study, 100 mmol·L^-1 is the optimum Na Cl concentration for salt-tolerance screening and seven indexes including relative seedling rate could be targeted for effective screening of salt-tolerance in large sesame germplasm. In addition, 7 SNP loci peaks significantly associated with salt tolerance were identified and 21 candidate salt-tolerant genes were discovered.